Knockdown of CYP6CR2 and CYP6DE5 reduces tolerance to host plant allelochemicals in the Chinese white pine beetle Dendroctonus armandi.

Bark beetles rely on detoxifying enzymes to resist the defensive terpenoids of the host tree. Insect cytochrome P450 (CYPs) plays a key role in the detoxification of pesticides and plant allelochemicals. CYP6 family is unique to Insecta, and its biochemical function is basically related to the metabolism of exogenous substances. In this study, we sequenced and characterized the full-length cDNAs of two CYP6 genes from Chinese white pine beetle, Dendroctonus armandi. Spatiotemporal expression profiling revealed that the expression of CYP6CR2 and CYP6DE5 was higher in larval and adult stages of D. armandi than that in other developmental stages, and that two genes predominantly expressed in brain, midgut, fat body, Malpighian tubules or hemolymph. The expression of CYP6CR2 and CYP6DE5 was significantly induced after feeding on the phloem of Pinus armandii and exposure to six stimuli [(±)- α -pinene, (-)-α-pinene, (-)-ß-pinene, (+)-3-carene, (±)-limonene and turpentine]. Importantly, silencing CYP6CR2 and CYP6DE5 separately could increase the sensitivity, led to a significant reduction of the activity of P450, resulting a significant increase in adult mortality after treatment with terpenoids. The comprehensive results of this study showed that in the process of host selection and colonization, the functions of CYPs were mainly to hydrolyze the chemical defense of the host and degrade odor molecules. These findings may help to develop new treatments to control this important pest.

Functional Characterization of Allatostatin C (PISCF/AST) and Juvenile Hormone Acid O-Methyltransferase in Dendroctonus armandi.

Allatostatin C (PISCF/AST) is a neuropeptide gene that affects juvenile hormone (JH) synthesis in the corpora allata. Juvenile hormone acid O-methyltransferase (JHAMT) is a key gene in the JH biosynthetic pathway. In this study, two genes encoding DaAST and DaJHAMT were cloned. Both DaAST and DaJHAMT were expressed in the larvae, pupae and adults of Chinese white pine beetle (Dendroctonus armandi), and highly expressed in the head and the gut. The expression of the two genes was induced by JH analog (JHA) methoprene and the functions of the two genes were then investigated by RNAi. Considering the role of hormones in metamorphosis, JHA significantly induced DaAST and DaJHAMT in the larval stage. DaAST knockdown in larvae, pupae and adults significantly increased the DaJHAMT mRNA levels. Moreover, knockdown of DaAST instead of DaJHAMT increased pupae mortality and the abnormal rate of emergence morphology and reduced emergence rates. However, knockdown of DaJHAMT instead of DaAST significantly reduced frontalin biosynthesis in adult males. The results showed that DaAST acts as an allatostatin and inhibits JH biosynthesis, and that JHAMT is a key regulatory enzyme for JH synthesis in the D. armandi.

Function of mevalonate pathway genes in the synthesis of frontalin in Chinese white pine beetle, Dendroctonus armandi (curculionidae: Scolytinae).

The Chinese white pine beetle (Dendroctonus armandi Tsai and Li) is a significant pest of pine forests in the Qinling and Bashan Mountains of China. Adult males commonly produce frontalin using precursors synthesized through the mevalonate pathway, which is regulated by juvenile hormone III (JHIII). In this study, the expression levels of mevalonate pathway genes were quantified after phloem feeding and topical application of the JHIII solution. The frontalin was quantified by gas chromatography-mass spectrometry. Both the phloem feeding and JHIII treatments produced an evident upregulation in the male gut, mainly in 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS) and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR). Moreover, HMGS, HMGR, isopentenyl diphosphate isomerase, and geranyl diphosphate synthase/farnesyl diphosphate synthase were upregulated in fed and JHIII-stimulated males of D. armandi under both conditions (solitary and paired). The expression levels were higher in paired compared to solitary males. Males had higher expression levels compared with females. Correspondingly, the phloem-feeding males produced more frontalin than JHIII-treated males, and the production of frontalin was higher in paired males than in solitary males. The knockdown of mevalonate pathway genes using RNAi in vivo effectively reduced the messenger RNA level of these genes and inhibited the production of frontalin. Among them, the silencing of HMGR or HMGS genes reduced the synthesis of frontalin most significantly.

Isolation and expression of five genes in the mevalonate pathway of the Chinese white pine beetle, Dendroctonus armandi (Curculionidae: Scolytinae).

The Chinese white pine beetle Dendroctonus armandi (Tsai and Li) is a significant pest of the Qinling and Bashan Mountains pine forests of China. The Chinese white pine beetle can overcome the defences of Chinese white pine Pinus armandi (Franch) through pheromone-assisted aggregation that results in a mass attack of host trees. We isolated five full-length complementary DNAs encoding mevalonate pathway-related enzyme genes from the Chinese white pine beetle (D. armandi), which are acetoacetyl-CoA thiolase (AACT), geranylgeranyl diphosphate synthase (GGPPS), mevalonate kinase (MK), mevalonate diphosphate decarboxylase (MPDC), and phosphomevalonate kinase (PMK). Bioinformatic analyses were performed on the full-length deduced amino acid sequences. Differential expression of these five genes was observed between sexes, and within these significant differences among topically applied juvenile hormone III (JH III), fed on phloem of P. armandi, tissue distribution, and development stage. Mevalonate pathway genes expression were induced by JH III and feeding.

Illuminating the Characteristics and Assembly of Prokaryotic Communities across a pH Gradient in Pit Muds for the Production of Chinese Strong-Flavor Baijiu.

Pit mud (PM), as an important source of microorganisms, is necessary for Chinese strong-flavor baijiu (CSFB) production. Although it has been revealed that the PM prokaryotic community diversities are influenced by its quality, product area, ages, etc., the characteristics and assembly process of the prokaryotic community in PMs across a pH gradient are still unclear. In this study, the regular changes of α- and ß-diversities of the prokaryotic community across a pH gradient in PMs were revealed, which could be divided into "stable", "relatively stable", and "drastically changed" periods. A total of 27 phyla, 53 classes, and 381 genera were observed in all given samples, dominated by Firmicutes, Bacteroidetes, Proteobacteria, Lactobacillus, Caproiciproducens, Proteiniphilum, etc. Meanwhile, the complexity of the network structure of the prokaryotic microbial communities is significantly influenced by pH. The community assembly was jointly shaped by deterministic and stochastic processes, with stochastic process contributing more. This study was a specialized report on elucidating the characteristics and assembly of PM prokaryotic communities across a pH gradient, and revealed that the diversity and structure of PM prokaryotic communities could be predictable, to some degree, which could contribute to expanding our understanding of prokaryotic communities in PM.

Identification and functional characterization of the sulfakinin and sulfakinin receptor in the Chinese white pine beetle Dendroctonus armandi.

The sulfakinin (SK) is an important signal molecule. As a neuromodulator, it mediates a variety of behavioral processes and physiological functions in invertebrates through the interaction with G-protein-coupled receptors (GPCRs). However, there is no report on the functional role of SK in the Chinese white pine beetle, Dendroctonus armandi. We have cloned and characterized SK and SKR genes in the D. armandi and carried out bioinformatics predictions on the basis of the deduced amino acid sequences, which are very similar to those from Dendroctonus ponderosa. The expression levels of the two genes were different between male and female adults, and there were significant changes in different developmental stages, tissues, and between starvation and following re-feeding states. Additionally, RNA-interference (RNAi) using double-stranded RNA to knock down SK and SKR reduced the transcription levels of the target genes and increased their body weight. In parallel, injection of SK caused a significant reduction in body weight and increase in mortality of D. armandi and also led to an increase in trehalose and a decrease in glycogen and free fatty acid. The results show that the SK signal pathway plays a positive and significant role in feeding regulation and provides a potential molecular target for the control of this pest.

Roles of Krüppel Homolog 1 and Broad-Complex in the Development of Dendroctonus armandi (Coleoptera: Scolytinae).

In insects, metamorphosis is controlled by juvenile hormone (JH) and 20-hydroxyecdysone (20E). Krüppel homolog 1 (Kr-h1), a key JH-early inducible gene, is responsible for the suppression of metamorphosis and the regulation of the Broad-Complex (Br-C) gene, which is induced by 20E and functions as a "pupal specifier". In this study, we identified and characterized the expression patterns and tissue distribution of DaKr-h1 and DaBr-C at various developmental stages of Dendroctonus armandi. The expression of the two genes was induced by JH analog (JHA) methoprene and 20E, and their functions were investigated by RNA interference. DaKr-h1 and DaBr-C were predominantly expressed in the heads of larvae and were significantly downregulated during the molting stage. In contrast, the DaKr-h1 transcript level was highest in the adult anterior midgut. DaBr-C was mainly expressed in female adults, with the highest transcript levels in the ovaries. In the larval and pupal stages, both JHA and 20E significantly induced DaKr-h1, but only 20E significantly induced DaBr-C, indicating the importance of hormones in metamorphosis. DaKr-h1 knockdown in larvae upregulated DaBr-C expression, resulting in precocious metamorphosis from larvae to pupae and the formation of miniature pupae. DaKr-h1 knockdown in pupae suppressed DaBr-C expression, increased emergence, caused abnormal morphology, and caused the formation of small-winged adults. These results suggest that DaKr-h1 is required for the metamorphosis of D. armandi. Our findings provide insight into the roles of DaKr-h1 and DaBr-C in JH-induced transcriptional repression and highlight DaKr-h1 as a potential target for metamorphosis suppression in D. armandi.

Identification and Functional Characterization of Antifreeze Protein and Its Mutants in Dendroctonus armandi (Coleoptera: Curculionidae: Scolytinae) Larvae Under Cold Stress.

Dendroctonus armandi (Tsai and Li) (Coleoptera: Curculionidae: Scolytinae) is considered to be the most destructive forest pest in the Qinling and Bashan Mountains of China. Low winter temperatures limit insect's populations, distribution, activity, and development. Insects have developed different strategies such as freeze-tolerance and freeze-avoidance to survive in low temperature conditions. In the present study, we used gene cloning, real-time polymerase chain reaction (PCR), RNA interference (RNAi), and heterologous expression to study the function of the D. armandi antifreeze protein gene (DaAFP). We cloned the 800 bp full-length cDNA encoding 228 amino acids of DaAFP and analyzed its structure using bioinformatics analysis. The DaAFP amino acid sequence exhibited 24-86% similarity with other insect species. The expression of DaAFP was high in January and in the larvae, head, and midgut of D. armandi. In addition, the expression of DaAFP increased with decreasing temperature and increasing exposure time. RNAi analysis also demonstrated that AFP plays an important role in the cold tolerance of overwintering larvae. The thermal hysteresis and antifreeze activity assay of DaAFP and its mutants indicated that the more regular the DaAFP threonine-cystine-threonine (TXT) motif, the stronger the antifreeze activity. These results suggest that DaAFP plays an essential role as a biological cryoprotectant in overwintering D. armandi larvae and provides a theoretical basis for new pest control methods.

Glide Mobility of a-Type Edge Dislocations in Aluminum Nitride by Molecular Dynamics Simulation.

Classical molecular dynamics simulations are performed to investigate the motion of a-type edge dislocations in wurtzite aluminum nitride (AlN). The nucleation and propagation of kinks are observed via the dislocation extraction algorithm. Our simulation results show that the nucleation energy of the kink pair in AlN is 1.2 eV and that the migration energy is 2.8 eV. The Peierls stress of the 1/3⟨112̅0⟩{101̅0} edge dislocation at 0 K is 15.9 GPa. The viscous motion of dislocations occurs when τ > τ p , and the dislocation velocity is inversely proportional to the temperature and directly proportional to the applied stress. Below room temperature, the value of the critical resolved shear stress (CRSS) on the prismatic plane is the lowest, which suggests that the dislocation mobility on the prismatic plane is the easiest. The CRSS on the pyramidal plane is always the highest at all temperatures, which suggests that pyramidal slip is the hardest among these three slip systems.

Identification of the Short Neuropeptide F and Short Neuropeptide F Receptor Genes and Their Roles of Food Intake in Dendroctonus armandi.

The short neuropeptide F (sNPF) is an essential signaling molecule that is evolutionarily conserved and involved in a broad range of physiological functions in the invertebrates, by interacting with sNPF receptors, which belong to G protein-coupled receptors (GPCR). However, the function of sNPF in regulating the food intake of Dendroctonus armandi has been unclear. In this study, we cloned and characterized cDNAs encoding sNPF and sNPF receptor in the D. armandi and made bioinformatics predictions on the deduced amino acid sequences. They had a high degree of similarity to that of Dendroctonus ponderosa. Quantitative real-time reverse transcription PCR (qRT-PCR) revealed that the transcript levels of both sNPF and sNPFR varied across developmental stages and body parts. In addition, the sNPF and sNPFR expression levels were upregulated in starved beetles, and the expression levels recovered after re-feeding. Furthermore, RNAi knockdown by the injection of sNPF and sNPFR dsRNA into beetles significantly increased mortality and reduced their food intake and body weight, and also caused decrease of glycogen and free fatty acid and increase of trehalose. These results indicate that sNPF signaling pathway plays an important role in the regulation of food intake and provides a potential molecular target for the eco-friendly control strategies of this pest.

Cloning and Expression of the Neuropeptide F and Neuropeptide F Receptor Genes and Their Regulation of Food Intake in the Chinese White Pine Beetle Dendroctonus armandi.

Neuropeptide F (NPF) is an important signaling molecule that acts as a neuromodulator to regulate a diversity of physiological and behavioral processes from vertebrates to invertebrates by interaction with NPF receptors, which are G protein-coupled receptors (GPCR). However, nothing is known about NPF in Chinese white pine beetle, Dendroctonus armandi, a destructive pest of natural and coniferous forests in the middle Qinling Mountains of China. We have cloned and characterized cDNAs encoding one NPF precursor and two NPF receptors in D. armandi and made bioinformatics predictions according to the deduced amino acid sequences. They were highly similar to that of Dendroctonus ponderosa. The transcription levels of these genes were different between larvae and adults of sexes, and there were significant differences among the different developmental stages and tissues and between beetles under starvation and following re-feeding states. Additionally, downregulation of NPF and NPFR by injecting dsRNA into beetles reduced their food intake, caused increases of mortality and decreases of body weight, and also resulted in a decrease of glycogen and free fatty acid and an increase of trehalose. These results indicate that the NPF signaling pathway plays a significant positive role in the regulation of food intake and provides a potential target for the sustainable management of this pest.

Isolation, Expression Profiling, and Regulation via Host Allelochemicals of 16 Glutathione S-Transferases in the Chinese White Pine Beetle, Dendroctonus armandi.

The Chinese white pine beetle (Dendroctonus armandi) is undoubtedly one of the most important pests causing ecological damage in the Qinling Mountains. When bark beetles invade conifers, they must overcome host tree defenses, including primary resistance and induced resistance responses. Moreover, this induced resistance occurs following herbivory by bark beetles. Bark beetles have a corresponding defense mechanism for degrading toxic compounds, and glutathione S-transferases (GSTs) can catalyze the binding of endogenous substances that reduce glutathione (GSH) to various harmful electrophilic substrates, increasing their solubility and facilitating their excretion from cells. In this experiment, we successfully obtained sixteen full-length sequences of D. armandi, which belonged to four GST categories (delta, epsilon, sigma, and theta). The transcript levels of sixteen GSTs in D. armandi were compared at four developmental stages (larvae, pupae, teneral adults, and adults), three different tissues (antennae, gut, and reproductive organs), and under various levels of terpenoid stimuli during feeding on phloem tissue to evaluate the various relevant modes of action. This study aids in the understanding of the interaction between monoterpenes and beetles, and beetles' detoxification through GSTs.

Isolation of CarE genes from the Chinese white pine beetle Dendroctonus armandi (Curculionidae: Scolytinae) and their response to host chemical defense.

BACKGROUND: Bark beetles rely on detoxifying enzymes to resist the defensive terpenoids of their host trees. Research on carboxylesterases (CarEs) has focused on their multiple functions in the metabolic detoxification of pesticides and plant allelochemicals, drug resistance, and juvenile hormone and pheromone degradation. RESULT: We identified eight new CarE genes in the Chinese white pine beetle (Dendroctonus armandi) and carried out bioinformatics analysis on the deduced full-length amino acid sequences. Differential transcript levels of CarE genes were observed between sexes; within these levels, significant differences were found among the different development stages, and between insects fed on the phloem of Pinus armandi and exposed to five stimuli [(-)-α-pinene, (-)-ß-pinene, (+)-3-carene, limonene and turpentine] at 8 and 24 h. CONCLUSION: Transcription levels of CarE genes suggest some relationship with the detoxification of terpenoids released by host trees. The functions of bark beetle esterase are mainly in hydrolyzing the host chemical defense and degrading odorant molecules during host selection and colonization. © 2018 Society of Chemical Industry.

Identification, Expression Patterns and RNA Interference of Aquaporins in Dendroctonus armandi (Coleoptera: Scolytinae) Larvae During Overwintering.

The ability to survive annual temperature minima could be a key determinant of distribution limits for insects under global climate change. Recent studies have suggested that insect aquaporins are indispensable for cellular water management under conditions that lead to dehydration and cold stress. Aquaporins are integral membrane water channel proteins in the major intrinsic protein superfamily and promote selected solutes and the movement of water across biological membranes. We cloned and characterized nine full-length aquaporins from Dendroctonus armandi (DaAqps), the most destructive forest pest in the Qinling Mountains of Shaanxi Province, China. Eight of the DaAqps belong to three classical aquaporin grades, including the Drosophila integral protein, the Pyrocoelia rufa integral protein, the entomoglyceroporins and one that belongs to the unorthodox grade of aquaporin 12-like channels. The DaAqps were increasingly expressed during different developmental stages and in different larval tissues, and expression peaked in mid-winter. They were tested under cold conditions for different lengths of time, and the expression of almost all DaAqps was down regulated with decreasing temperatures and long-term exposure to cold conditions. However, when the lowest temperatures were reached, the levels were immediately upregulated. These genes indicate that cold tolerance can improve through mortality responses at low temperatures after RNA interference of DaAqps. In our study, we analyzed the molecular response, expression patterns, and RNA interference of DaAqps and clarified the crucial role of protective compounds (aquaporins) underlying D. armandi cold tolerance and provide a new pest control method.

Identification, Expression Patterns, and Functional Characterization of Chemosensory Proteins in Dendroctonus armandi (Coleoptera: Curculionidae: Scolytinae).

The Chinese white pine beetle, Dendroctonus armandi Tsai and Li (Coleoptera: Curculionidae: Scolytinae), is a serious pest of coniferous forests in China. Thus, there is considerable interest in developing eco-friendly pest-control methods, with the use of semiochemicals as a distinct possibility. Olfaction is extremely important for fitness of D. armandi because it is the primary mechanism through which the insect locates hosts and mates. Thus, here we characterized nine full-length genes encoding chemosensory proteins (CSPs) from D. armandi. The genes were ubiquitously and multiply expressed across different developmental stages and adult tissues, indicating various roles in developmental metamorphosis, olfaction, and gustation. Ligand-binding assays implied that DarmCSP2 may be the carrier of D. armandi pheromones and various plant host volatiles. These volatiles were identified through RNA interference of DarmCSP2 as: (+)-α-pinene, (+)-ß-pinene, (-)-ß-pinene, (+)-camphene, (+)-3-carene, and myrcene. The systematic chemosensory functional analysis of DarmCSP2 in this study clarified the molecular mechanisms underlying D. armandi olfaction and provided a theoretical foundation for eco-friendly pest control.